Oxygen therapy of neonatal respiratory distress syndrome increases the production of highly reactive partially reduced oxygen molecules within the lung. These reactive oxygen species can exceed antioxidant defenses, resulting in lung injury which is often exacerbated in premature infants due to a gestational-age dependent deficiency in antioxidant defenses. The long-term objective of these studies is to determine the molecular regulation of antioxidant gene expression during fetal lung development. The specific aim of this proposal is to determine the developmental regulation of pulmonary ceruloplasmin gene expression. Ceruloplasmin is a copper binding acute-phase plasma protein which can scavenge free radicals, prevent lipid perioxidation and provide tissue copper for Cu/Zn superoxide dismutases. Although ceruloplasmin is thought to be exclusively liver-derived, preliminary studies in our laboratory reveal that 1) during fetal development the ceruloplasmin gene is expressed in human and rat lung tissue 2) the induction of pulmonary ceruloplasmin gene expression during fetal development occurs late in gestation, accompanied by expression of Cu/Zn superoxide dismutase and a differentiation of Type II cells and 3) hyperopia increases the pulmonary ceruloplasmin gene expression seen in fetal and newborn rats and mediates a tissue-specific induction of pulmonary ceruloplasmin gene expression in adult animals. To examine these findings in detail, we propose to clone the rat ceruloplasmin gene, determine the nucleotide sequence and identify potential promoter and enhancer elements. The cis-acting DNA sequences mediating tissue-specific pulmonary ceruloplasmin gene expression will then be localized using deletional analysis and cell-type specific transfection studies. DNAse footprinting and DNA-protein binding assays will be employed to identify cell-specific trans-acting regulatory factors. The cells responsible for pulmonary ceruloplasmin production will be identified by in-situ hybridization and tissue culture of these cell types will be established. Finally, ceruloplasmin gene transcription, mRNA abundance and protein synthesis and secretion in response to a variety of hormones and growth factors will be examined in lung cell tissue cultures to determine the factors which mediate developmental tissue specific pulmonary ceruloplasmin gene expression. Understanding the molecular determinants of antioxidant gene expression during development may eventually allow manipulation of gene expression in the developing human fetus at risk for premature birth.